Microfluidic device for cell culture

ABSTRACT

A microfluidic cell culture apparatus includes a cell retention chamber and a perfusion channel. The cell retention chamber has a structured surface. The structured surface includes a major surface from which a plurality of projections extends into the chamber. The plurality of projections are arranged to suspend cells cultured in the chamber above the major surface. The first perfusion channel is configured to provide laminar flow of a fluid through the channel and forms a plurality of openings in communication with the cell retention chamber. The openings are configured to prevent cells from the retention chamber from entering the perfusion channel.

CLAIMING BENEFIT OF PRIOR FILED U.S. APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/250,754, filed on Oct. 12, 2009. The content of this document andthe entire disclosure of publications, patents, and patent documentsmentioned herein are incorporated by reference.

FIELD

The present disclosure relates to apparatus for culturing cells; moreparticularly to microfluidic cell culture apparatuses.

BACKGROUND

Cells cultured on flat cell culture ware often provide artificialtwo-dimensional sheets of cells that may have significantly differentmorphology and function from their in vivo counterparts. Cultured cellsare important to modern drug discovery and development and are widelyused for drug testing. However, if results from such testing are notindicative of responses from cells in vivo, the relevance of the resultsmay be diminished. Cells in the human body experience three dimensionalenvironments completely surrounded by other cells, membranes, fibrouslayers, adhesion proteins, etc. Thus, cell culture apparatuses thatbetter mimic in vivo conditions and that prompt cultured cells to havein vivo-like morphology and function are desirable.

Much progress has been made in cell culture configuration and systems tobetter mimic in vivo conditions and maintain differentiated cells, suchas hepatocytes, in culture for longer periods of time. For example,collagen sandwich culture systems, 3D cell culture, and microfluidicperfusion systems have provided some enhancement in cell performancerelative to conventional cultures devices in maintaining viable cellcultures with some phenotypic relevance. Other methodologies that havebeen used to prolong cell viability and function include the use ofmodified cell culture media, co-cultures, and the use of variousextracellular matrices to promote 3D cellular organization. However,mimicking complex in vivo microenvironment that modulates cellularfunction for successful long-term cultures of cells remains a challenge.Accordingly, even with such advances, limited improvement in cellcultured cell function has been achieved.

BRIEF SUMMARY

The present disclosure describes, among other things, microfluidicdevices that provide dynamic cell culture conditions via multipleperfusion channels and virtual suspension of cells on structuredsupports or encapsulated by structured supports. The devices may mimicthe architecture, perfusion and flow of tissue in vivo and allow forformation of tissue-like structure and morphology. For example, in theExamples provided below, hepatocytes cultured on devices describedherein showed restored membrane polarity that was extended in threedimensions, formation of bile canaliculi structures, and transportfunction without the addition of biological or synthetic matrices orcoagulants.

The devices described herein have a perfusion channel through which cellculture medium or other fluid compositions may be flowed. The perfusionchannel is in fluid communication with a cell retention chamber in whichcells may be cultured. The cell retention chamber includes a structuredsurfaces configured to prevent cell spreading, which may promotethree-dimensional cell morphology. The structured surfaces includeprojections configured to suspend cells above the bottom of thestructured surface. The surface area of the surface of the projectionswith which the cells come into contact, in many embodiments, is lessthan the contact surface area of a cell to be cultured in the device(i.e., less than the surface area of a cell that would come into contactwith a flat, non-structured surface). By limiting the surface area thatthe cells may contact, cell spreading may be inhibited andthree-dimensional cell morphology may be promoted. In some instances,the structured surface can promote or retain cell polarity, such aspolarity of hepatocytes.

The structured surfaces may form one or more troughs through which fluidmay flow. The bottom of the troughs may be formed by the bottom of thestructured surface and the sides of the projections may form the sidesof the trough. In various embodiments, the microfluidic culture deviceshave an inlet and outlet in fluid communication with one or more troughsof the structured surfaces that allow fluid to be introduced into orremoved from the troughs. In situations where the cultured cells formtight cell-cell junctions (e.g., adopt tissue like morphology), thecells may fluidly isolate the troughs and the perfusion channel,allowing independent gradients to be formed across the cell chamber. Inaddition, the trough(s) and perfusion channel can be effectively used tosimulate multidirectional flow in vivo. In some cases, the cariousgradients that may result or the multidirectional flow may encouragecell polarity.

The devices and methods described herein may provide one or moreadvantages over prior microfluidic or other culture devices. Forexample, embodiments of the devices described herein may providestructural design to enable 3D tissue-like organization of cells andrestoration of in vivo-like membrane polarity, may provide sustainabledynamic in vivo-like conditions for long-term cell culture andcell-specific functionality in vitro for evaluation of toxicity(including chronic toxicity) and studies of drug-drug interaction (overlonger term), may provide dynamic cell culture conditions, such ascontrolled supply of oxygen and nutrients, oxygen gradient and shearstress control, and may allow for control of oxygen levels and nutrientsto mimic physiological conditions. The multiple flow channels provideefficient and effective transport of nutrients, removal of waste, andsupply of oxygen. The troughs and perfusion channel can be effectivelyused independently to generate gradients across the cell chamber andsimulate multidirectional flow in vivo. A perfusion regime that promotesthe restoration of polarity and extends a bile canalicular structure inthree dimensions can be realized. These and other advantages of thevarious embodiments of the devices and methods described herein will bereadily apparent to those of skill in the art upon reading thedisclosure presented herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic perspective view of an embodiment of amicrofluidic apparatus.

FIGS. 2A-C are schematic cross sections of embodiments of a microfluidicdevice of FIG. 1, taken through line c-c between lines a-a and b-b ofFIG. 1.

FIGS. 3A-B are schematic diagrams of structured surfaces without (A) andwith (B) cells disposed on the surface.

FIG. 4 is a schematic cutaway perspective view of an embodiment of amicrofluidic device.

FIG. 5 is a schematic top down view of a portion of an embodiment of amicrofluidic device.

FIG. 6 is a schematic cross-section of the device shown in FIG. 5 takenalong line 6-6.

FIG. 7 is a schematic cross-section of the device shown in FIG. 5 takenalong line 7-7.

FIG. 8 is a schematic diagram of the device shown in FIG. 6 showingschematic cells cultured in the device.

FIGS. 9A-B are schematic top down views of portions of embodiments ofmicrofluidic devices.

FIG. 10 is a schematic cross-section of an embodiment of a microfluidicdevice.

FIGS. 11-12 are schematic top-down views of embodiments of devices.

FIGS. 13A-C are schematic top-down views of portions of microfluidicdevices showing examples of fluid flow.

FIGS. 14A-E show a schematic diagram of an embodiment of a microfluidicapparatus (A), magnified images of portions of a microfluidic apparatus(B-C), and images at higher magnification (D-E).

FIG. 15 is a schematic top down view of a portion of an embodiment of amicrofluidic apparatus.

FIGS. 16A-F are a schematic illustration of a photolithographic processfor preparing a device or portion thereof.

FIGS. 17A-B are schematic cross sections of microfluidic devices formedfrom top part and bottom parts.

FIGS. 18-19 are flow diagrams of overviews of methods.

FIG. 20 is an image of a silicon master used for forming a portion of amicrofluidic device.

FIG. 21 is an image of a replicated assembled device.

FIGS. 22A-B are images of alternative embodiments of devices, differentdimensions of retention posts and bottom channel (or top channel)substructures.

FIGS. 23A-B are fluorescent images showing fluid flow through amicrofluidic device.

FIGS. 24A-B are fluorescent images showing fluid flow through amicrofluidic device in which cells are cultured.

FIG. 25 is an image (20×) of human primary hepatocytes packed andcultured in a cell chamber of a microfluidic device for 7 days.

FIG. 26A-B are representative fluorescent images (A: 5×, B: 20×) showingthe results of live/dead staining of hepatocytes cultured in a cellchamber of a microfluidic device after 7 days of incubation.

FIGS. 27A-B are fluorescent and brightfield images of hepatocytes packedin 3D in a cell chamber of a microfluidic device without bottomsubstructures (22A) and with bottom substructures (22B) taken after 7days of incubation. In FIG. 22A, the left most panel is a fluorescentimage (5×) and the three rightmost panels are brightfield images at 5×,20× and 20× (left to right, respectively). In FIG. 22B, the left mostpanel is a fluorescent image (10×), the middle panel is a brightfieldimage (10×), and the right panel is a brightfield image (20×).

FIG. 28 shows three brightfield images of cells cultured in amicrofluidic device under perfusion conditions. In the left panel, cellsare shown in an assembled device. In the middle panel, cells are shownafter the device cover was removed. In the right panel, cells are shownafter being dislodged from the device.

FIG. 29 shows three brightfield images of cells cultured under staticconditions. In the left panel, cells are shown in an assembled device.In the middle panel, cells are shown after the device cover was removed.In the right panel, cells are shown after being dislodged from thedevice.

FIG. 30 shows two brightfield images of cells cultured on a 96 wellplate having the structured bottom surface of the microfluidic devicereplicated on the bottom surface of the well. The image on the left isof cells cultured on plasma treated PDMS substrate. The image on theright is on cells cultured on non-treated PDMS.

FIG. 31 shows fluorescent images of MRP2 protein immunostained humanprimary hepatocytes cultured in a conventional 96 well plate (leftpanel), and in a microfluidic device (middle and right panels).

FIG. 32 is a fluorescent image of connexin-32 protein immunostainedhuman primary hepatocytes cultured in a microfluidic device.

FIGS. 33A-B are images of a fluorescine diacetate transport functionassay for MRP2 hepatocyte transporter of cells cultured in aconventional 96 well plate (A) and in a microfluidic device (B).

The schematic drawings presented herein are not necessarily to scale.Like numbers used in the figures refer to like components, steps and thelike. However, it will be understood that the use of a number to referto a component in a given figure is not intended to limit the componentin another figure labeled with the same number. In addition, the use ofdifferent numbers to refer to components is not intended to indicatethat the different numbered components cannot be the same or similar.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings that form a part hereof, and in which are shown byway of illustration several specific embodiments of devices, systems andmethods. It is to be understood that other embodiments are contemplatedand may be made without departing from the scope or spirit of thepresent disclosure. The following detailed description, therefore, isnot to be taken in a limiting sense.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise.

As used in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

As used herein, “have”, “having”, “include”, “including”, “comprise”,“comprising” or the like are used in their open ended sense, andgenerally mean “including, but not limited to.”

Any direction referred to herein, such as “top,” “bottom,” “left,”“right,” “upper,” “lower,” “above,” below,” and other directions andorientations are described herein for clarity in reference to thefigures and are not to be limiting of an actual device or system or useof the device or system. Devices or systems as described herein may beused in a number of directions and orientations.

As used herein, “structured surface” means a surface having apredetermined topography. A structured surface includes a major surfaceand projections extending from the major surface, which define thepredetermined topography. The projections have a surface facing in adirection substantially the same as the major surface, and thesesurfaces of the projections, along with the exposed portions of themajor surface (those portions on which no projection lies), comprise the“structured surface”.

As used herein, a “trough”, as it relates to a structured surface, meansa depression or channel formed along the major surface of the structuredsurface between at least two projections extending from the majorsurface. In many embodiments, a “trough” is a continuous path thatextends along the length of the major surface of the structured surface.A trough may take any path along the major surface and is defined, atleast in part, by the projections extending from the major surface.

As used herein, “suspend”, as it related to cells relative to a surface,means “to support the cell above the surface.”

As used herein, a “perfusion channel”, as it relates to a microfluidicdevice for culturing cells, means a channel through which a cell culturemedium may flow, which channel is configured to allow the cell culturemedium to perfuse cells cultured in the device. Typically, a perfusionchannel is configured to provide laminar flow (i.e., non-turbulent flow)of the cell culture medium and forms openings through which the cellculture medium may pass to perfuse the cells.

The present disclosure describes, among other things, microfluidicdevices that provide dynamic cell culture conditions via multipleperfusion channels and virtual suspension of cells on structuredsupports or encapsulated by structured supports. The devices may mimicthe architecture, perfusion and flow of tissue in vivo, allowing forcultured cells to adopt in vivo-like morphology and functionality. Suchdevices, as described herein below, have been shown to promote andmaintain 3D in vivo-like cellular structure that promotes cell-cellsignaling, the restoration of polarity in three dimensions, andcell-specific functionality in vitro for long term cell culture withoutaddition of biological or synthetic matrices or coagulants.

In various embodiments, such devices have multiple inlets and outletsfor independent flow as well as for seeding or infusing cells into aready-to-use (no assembly required) device. The devices may allow forcontrol of perfusion to provide optimized physiologically relevant cellculture conditions, such as media flow and shear stress similar totissue microcirculation, oxygen and growth media supply, and removal ofwaste components, catabolites and metabolites. The devices may also havemultiple perfusion channels where there are two parallel flow channelson either side of the cell chamber and there is a lower flow channelthat supports independent flow and virtual suspension of cells onstructured supports (pillars or channel sub-structures) at the bottom ofthe cell chamber. Such devices may be used for testing drug candidateson target cells, drug-drug interactions, metabolism and toxicity of newdrug candidates as well as their metabolites, and transport of drugcandidates and their metabolites. The devices may be arrayed andpackaged in format for throughput to perform screening assays, such astoxicity screening. In various embodiments, the devices are configuredto be fitted (or are fitted) with biosensors to monitor environmentalconditions such as O₂, CO₂, flow rates, pH and temperature.

Referring now to FIG. 1, a schematic perspective view of an embodimentof microfluidic cell culture apparatus 100 is shown. The depictedapparatus 100 has an inlet 330 and an outlet 335 in communication withan internal cell retention chamber (not show in FIG. 1). The depictedapparatus also has an inlet 310 and an outlet 315 in fluid communicationwith a first internal perfusion channel (not show in FIG. 1) and aninlet 312 and an outlet 317 in fluid communication with a secondinternal perfusion channel (not show in FIG. 1). It will be understoodthat a cell culture apparatus 100 as described herein may include anynumber of inlets 310, 312 and outlets 315, 317 and any number ofperfusion channels. It will be further understood that an inlet 310,312, as depicted, may be an outlet, and an outlet 315, 317, as depicted,may be an inlet, depending on the direction of fluid flow. One inlet orone outlet is in fluid communication with more than one perfusionchannel.

Referring now to FIGS. 2A-C, schematic cross sections of variousembodiments of the apparatus 100 of FIG. 1, taken along line c-c betweenlines a-a and b-b as shown in FIG. 1, are shown. The cell retentionchamber of the apparatus 100 includes a structured surface 140 on whichcells may be cultured. The structure surface 140 includes a majorsurface 149 (bottom surface in the depicted figures) facing the cellretention chamber. A plurality of projections 144 extend from the majorsurface 149 into the cell retention chamber. As illustrated in thedepicted embodiments, the projections 144 may be arranged in anysuitable manner and may be of any suitable shape or configuration. Inthe embodiments depicted in FIGS. 2A-2B, the projections 144 are in theform of pillars. While the pillars shown in FIGS. 2A-B are shown ashaving a rectangular cross-sectional shape, it will be understood thatthe pillars may have any suitable cross-sectional shape, such ascircular, ellipsoid, hexagonal, triangular, v-shaped, orirregular-shaped, or the like). In the embodiment shown in FIG. 2C, theprojections 144 form ridges extending the length of the structuredsurface 140. While the ridges depicted in FIG. 2C are linear, it will beunderstood that the ridges be of any suitable shape includingsinusoidal, serpentine, irregular, or the like.

Referring now to FIGS. 3A-C, schematic views of a structured surface 140of a cell retention chamber, without (3A) and with (3B) cells 200cultured on the surface 140 are shown. The structured surface 140 isconfigured to restrict spreading of cells 200 cultured on the surface.This can be accomplished by minimizing the surface area of thestructured surface 140 that contacts the cells when the cells 200 arecultured in the cell retention chamber of the apparatus. In general, theprojections 144 have a diametric dimension d, such as a width, diameter,or the like, that is less than a diametric dimension of a cell to becultured in the chamber. For example, the diametric dimension d of acell contacting surface 146 (the top surface in the depicted embodiment,which is generally opposing the surface in contact with or extendingfrom the major surface 149) of a projection 144 may be about half thediametric dimension of cells 200 to be cultured in the apparatus. Invarious embodiments, the diametric dimension d of the cell contactingsurface 146 of the projection 144 is between about 5 micrometers andabout 15 micrometers or between about 5 micrometers and 10 micrometers.When projections 144 are pillars (as opposed to elongate ridges as shownin FIG. 2C), the surface area of the cell contacting surface 146 may beless than the surface area of a cell that would come into contact with aflat, non-structured surface. For example, the surface area of the cellcontacting surface 146 may be one half, one quarter, or less than onquarter of the surface are of a cell that would come into contact with aflat, non-structured surface. In various embodiments, the surface areaof the cell contacting surface 146 is between about 25 squaremicrometers and 225 square micrometers.

Further, the projections 144 are spaced apart such that the distance Dbetween neighboring projections 144 is sufficiently small to prevent acell from contacting the major surface 149 of the structured surface. Inthis manner, the projections 144 suspend the cells 200 above the majorsurface 149, and the surface area of the structured surface 140 that thecells 200 may come into contact with is limited to the cell contactingsurfaces 146 of the projections 144. The distance D between neighboringprojections 144 may be any suitable distance, such as less than thediametric dimension of an average cell 200 to be cultured in theapparatus or less than half the diametric dimension of an average cell200 to be cultured in the apparatus. In various embodiments, thedistance D between neighboring projections 144 is between about 5micrometers and about 15 micrometers or between about 5 micrometers and10 micrometers.

The projections 144 may extend from the major surface 149 of thestructured surface 140 any suitable distance such that the cells 200 areeffectively suspended above the major surface 149. For example, theprojections may have a height h of greater than about 5 micrometers. Invarious embodiments, the projections 144 have a height h of betweenabout 5 micrometers and about 25 micrometers.

The projections 144 of the structured surface 140 may be arranged in aregular or irregular pattern. For purposes of manufacturing, theprojections 144 are arranged in a regular pattern or an array.Projections in an array may be of the same or difference shape,dimension, or configuration.

Referring now to FIGS. 4-8, various views of embodiments of microfluidicdevices 100 are shown. FIG. 4 is a schematic cutaway perspective view ofan embodiment of a microfluidic device 100; FIG. 5 is a schematic topdown view of a portion of an embodiment of a microfluidic device 100;FIG. 6 is a schematic cross-section of the device shown in FIG. 5 takenalong line 6-6; FIG. 7 is a schematic cross-section of the device shownin FIG. 5 taken along line 7-7; and FIG. 8 is a schematic diagram of thedevice shown in FIG. 6 showing cells 200 cultured in the device 100.

In the embodiments depicted in FIGS. 4-8, the devices 100 include a cellretention chamber 130 into which cells 200 may be placed for culture.The chamber 130 may be of any suitable size to allow culture of adesired number of cells 200. In various embodiments, the chamber 130 issized and configured to allow culture of two to six cells across thewidth and two to three cells across the height of the chamber. Having awidth of two to six cells and a height of two to three cells allows forready diffusion of nutrients or other agents from perfusion channels120, 122 to the cells cultured in the chamber. It will be understoodthat the size of cells may vary depending on cell type and cultureconditions. Accordingly, the appropriate size of the chamber 130 may bevaried to provide for the desired number of cell width and heightdimensions. It will also be understood that the size of cells of a givencell type under the same conditions may vary from cell to cell.Generally, when dimensions are described herein based on cell size, thedimensions are based on the average size of the cultured cells.

In various embodiments, the chamber 130 has a width of between about 80and 120 micrometers (about 4 to 5 times the diameter of a typical cell,which is between about 20 and 25 micrometers), such as about 105micrometers. The chamber 130 may be of any suitable height, e.g. betweenabout 30 micrometers and 80 micrometers, between 35 and 50 micrometers,or about 45 micrometers. The chamber 130 may be of any suitable length,e.g., between about 100 micrometers and about 30,000 micrometers,between about 150 micrometers and about 20,000 micrometers, or betweenabout 200 micrometers and about 15,000 micrometers. In many cases, achamber 130 having a shorter length is more likely to be effectivelypacked with cells (if desired) relative to chambers having longerlengths.

The chamber 130 has a structured surface 140 that forms one or moretroughs 142 between cell contacting surfaces 146. The troughs 142 areconfigured to provide channels for fluid retention or flow adjacentcells 200 cultured in the chamber 130. Accordingly, the troughs 142 areof a width that does not permit cells 200 cultured in the chamber 130 toblock flow of fluid through the trough 142. That is, the widths of thetroughs 142 are less than the width of the cells 200 to be cultured inthe chamber 130. For example, the troughs 142 may be half the width of acell 200 to be cultured. It will be understood that the width of thetrough(s) 142 may be varied depending on the size of the cells 200 to becultured in the apparatus 100. In various embodiments, the troughs havea width of less than about 15 micrometers, of between about 5micrometers and about 15 micrometers or between about 5 micrometers andabout 10 micrometers. In some embodiments, the troughs 142 havegenerally uniform widths along the length of the structured surface 140.In general, the troughs 142 are formed by the major surface (see, e.g.,reference numeral 149 of FIGS. 2A-C) of the structured surface 140 andthe sides of the projections 144. Accordingly, the troughs 142 may takeany pathway along the major surface of the structured surface 140,depending on the shape and configuration of the projections 144. Thetroughs 142 extend the length of the structured surface 140.

The troughs 142 may be used to carry or retain fluid compositions thatcan deliver agents to the cells 200 or remove agents from the cellchamber 130. By way of example a composition comprising nutrients, suchas a cell culture medium, may be placed in the troughs 142 to delivernutrients to the cultured cells 200 or to remove waste products from thecells. Agents to be tested, such as pharmacologic agent, may bedelivered to the cells 200 via the troughs 142. Agents that may inducecellular polarization, agents or compositions that mimic an in vivophysiological environment, or the like, may be introduced into thetroughs 142. In various embodiments, culture of cells 200 in the chamber130 fluidly isolates, at least partially, the troughs 142 from theperfusion channels 120, 122. For example, cells 200 cultured in thedevice 100 may interact to form a tissue-like morphology with cell-cellinteractions that can inhibit or reduce bulk movement of fluid betweenthe troughs 142 and the perfusion channels 120, 122

Still with reference to FIGS. 4-8, the depicted devices 100 includefirst 120 and second 122 perfusion channels through which fluid mayflow. In various embodiments (not shown), a device includes one or morethan two perfusion channels. The channel includes retention posts 160forming openings 150 that provide for fluid communication between thechamber 130 and the perfusion channels 120, 122, allowing diffusion ofagents between the perfusion channels 120, 122 and the cell chamber 130via the openings 150. The openings 150 are of dimensions to preventcells in the chamber 130 from entering a perfusion channel 120, 122. Forexample, the openings 150 may have a height, width or diametricdimension of less than about 20 micrometers, less than about 15micrometers, less than about 10 micrometers or about 5 micrometers.While shown in FIGS. 4-8 as having a rectangular cross-sectional shape,it will be understood that retention posts 160 may be of any suitableshape or configuration. In various embodiments, the retention posts 160have an ellipsoid, circular, triangular, w-shaped, or irregularcross-sectional shape, or the like.

The perfusion channels 120, 122 may be of any suitable dimension. Invarious embodiments, the height of a perfusion channel 120, 122 is thesame as the height of the cell retention chamber 130, and in someembodiments the height of a perfusion channel 120, 122 is the differentthan the height of the cell retention chamber 130. In embodiments, thefirst and second perfusion channels, 120, 122, are configured to carryfluid such as cell culture medium. In embodiments, the first and secondperfusion channels 120, 122 are configured to prevent cells fromentering the perfusion channels 120, 122 from the cell retention chamber130. In some embodiments, the height of a perfusion channel is betweenabout 30 micrometers and about 80 micrometers, between about 35 andabout 50 micrometers, or about 45 micrometers. It may be desirable forthe width of the perfusion channel to be greater than or equal to about1.5 times the diametric dimension of cells cultured in the device sothat if a cell happens to enter the perfusion channel, the cell willpass through the channel without blocking flow. In some embodiments, thewidth of a perfusion channel is between about 30 micrometers and about1000 micrometers, between about 30 micrometers and about 100micrometers, or between about 30 micrometers and about 45 micrometers.Typically, the perfusion chamber 120, 122 runs along side of the cellretention chamber 130 along the length of the chamber.

Referring now to FIGS. 9A-B, schematic top down views of portions ofembodiments of microfluidic devices 100 are shown. The devices 100 aresimilar to the device shown in FIG. 5, with like reference numbersreferring to like parts or components. Some of the projections 144 shownin FIG. 9A-B are square pillars rather than elongate projections asshown in FIG. 5 (of course, some of the projections in FIGS. 9A-B areelongate projections). The projections 144 depicted in FIGS. 9A-B format least a portion of a trough 142 that runs the length of thestructured surface 140. As shown by the line in FIG. 9B, the trough 142may be of any suitable shape and may be considered to take any suitablepath along the length of the structured surface 140. When at least someof the projections 144 allow for a trough 142 to take more than onepath, the trough 142 may be considered to take any suitable path.

Regardless of the structure of the trough(s) and projections, thestructured surface, in various embodiments, is configured to restrictthe spreading of cells, which may promote three-dimensional tissue-likemorphology and cell-cell interaction. The configuration of thestructured surface, and thus the shape and configuration of theprojections and path and configuration of the trough(s), may be varieddepending on the cells to be cultured so that the desired effects (e.g.,3D tissue-like morphology) are achievable. In general, it is desiredthat the projections 144 facilitate virtual suspension of cells culturedin the chamber. The projections 144 and the trough(s) 142 together mayfacilitate virtual suspension of cells, where the cells rest on top ofthe projections and fluid within the trough(s) assists in suspending thecells. By creating such virtual suspension, it is believed thatcontrolled cell aggregation or rearrangement of cells into tissue-likearchitecture can be promoted.

As shown in FIG. 10, the top and bottom surfaces of the cell retentionchamber may be structured surfaces 140, 140′. By providing structuredsurfaces 140, 140′ at both the top and bottom of the chamber 130interaction of the device 100 with cells cultured in the device can befurther minimized (relative to only one surface). In addition, the sidesurfaces of the chamber 130 are effectively formed by the retentionposts 160 and openings of the perfusion channels 120, 122, which areeffectively structure surfaces as well. When the chamber 130 is packedwith cultured cells, the top structured surface 140′ may form channels,along with the cells, through which fluid may flow. That is, cells inculture may form a seal between projections, forming a sealed channelthrough which fluid such as cell culture media may flow. In embodiments,these cell-formed sealed channels may allow for the delivery of fluid toone side of cells in culture, separate from the fluid delivered toanother side of cells in culture, thereby allowing cells to establishpolarity in culture, when they are exposed to a different fluid on oneside versus another side.

Referring now to FIGS. 11-12 schematic top-down views of embodiments ofdevices 100 are shown. Inlets 310, 312, 320, 330 and outlets 315, 317,325, 335 are shown. The inlets and outlets are accessible from theexterior of the device 100. A microfluidic device as described hereinmay include any suitable number of inlets and outlets. It will beunderstood that an inlet may serve as an outlet and an outlet may serveas an inlet, depending on the direction of fluid flow. In the embodimentdepicted in FIG. 11, the device 100 has a perfusion channel inlet 310, atrough inlet 320, a cell chamber inlet 330, a perfusion channel outlet315, a trough outlet 325, and a cell chamber outlet 335. The areadepicted in the dashed box in FIG. 11, is a schematic representation ofthe area of the device shown in, e.g., FIG. 5. The perfusion channelinlet 310 and outlet 315 are in fluid communication with the perfusionchannels 120, 122 (e.g., as depicted in FIG. 5) and allow fluid to enterthe inlet 310 flow through the perfusion channels 120, 122 and exit theoutlet 315. In the embodiment depicted in FIG. 12, inlet 310 and outlet315 are in fluid communication with one perfusion channel 120 (e.g., asdepicted in FIG. 5); and inlet 312 and outlet 317 are in fluidcommunication with another perfusion channel 122 (e.g., as depicted inFIG. 5). Thus, independent control of the content, rate, and directionof the flow within a perfusion channel can be achieved.

In the embodiments depicted in FIGS. 11-12, the trough inlet 320 andtrough outlet 325 are in fluid communication with one or more troughs142 (e.g., as depicted in FIG. 5). Of course, one or more inlets andoutlets may be employed if it is desired to independently control thecontent rate or direction of flow of fluid in a given trough or troughs.In the depicted embodiments, the cell chamber inlet 330 and outlet 335are in fluid communication with the cell chamber. Accordingly, dependingon the number of inlets and outlets provided, the composition, rate ordirection of flow may be varied as desired between perfusion channelsand troughs.

By way of example and with reference to FIGS. 13A-C, examples of flowthrough a representative microfluidic device are shown. The direction offlow is indicated by the arrows in the perfusion channels 120, 122 andthe troughs 142, 142′. The rate of flow is indicated by the length ofthe arrows. The same of different fluid compositions may be introducedinto perfusion channels 120, 122 and the troughs 142, 142′. In someembodiments, the direction of flow in a channel or trough may bechanged, the rate of flow may be changed, or the composition of thefluid flowing through a channel or trough may be changed at any desiredtime.

Pumps, syringes, or other suitable injection or infusion device may beemployed to introduce fluid into an inlet in communication with a cellchamber, trough or perfusion channel. The microfluidic devices describedherein can readily be adapted for use with available robotic fluidhandling systems.

The configuration of the structured surface(s) and the perfusionchannels, as well as the composition, direction, and rate of flow can bevaried in microfluidic devices, as described herein, as desired toachieve a suitable device that closely mimics in vivo tissue conditions.

In many embodiments, the flow through the perfusion channels or troughsis configured to be laminar, which as used herein means that thedirection of flow at any given point in the channel or trough isgenerally in the same direction. Alternatively, laminar flow, forpurposes of the present disclosure, can be considered as non-turbulent.Due to microfluidic nature of the device and pressure driven flows usedfor device perfusion pressure drops may develop along the perfusionchannels as well as trough. As the length of the chamber increases theflow resistance will increase, obstructing the independent flows introughs and perfusion channels. Thus the dimensions of the chamber mayvary depending on the desired flow characteristics. For example, in somesituations flow in a trough in a direction generally opposite that of aperfusion channel may be achievable across a chamber length of 500micrometers but may not be achievable across a chamber length of 1500micrometers.

Referring now to FIG. 14A-E, an embodiment of a microfluidic apparatusis shown. In the top panel (FIG. 14A) a schematic overview is shown; inthe middle panels (FIGS. 14B-C) magnified images of a representative areshown; and in the lower panels (FIGS. 14D-E) images at highermagnification are shown. In the depicted embodiment, the first 120 andsecond 122 perfusion channels extend beyond the cell culture chamber 130to the inlets 310, 312 and outlets 315, 317. The structured surface 140also extends beyond the cell chamber 130 to inlet 320 and outlet 325.Inlet 330 is in fluid communication with chamber 130 and provides accessfor introduction of cells into chamber 130. In the depicted embodiment,retention posts 131 are positioned at one end of the chamber to preventcells introduced into the chamber 130 from migrating past the retentionposts 131 towards outlet 335, which is also in fluid communication withthe chamber 130. It will be understood that an inlet may be an outletand an outlet may be an inlet, depending on the direction of fluid flow.

Referring now to FIG. 15, an alternative embodiment of a portion of amicrofluidic cell culture apparatus is shown. In the depictedembodiment, a linear trough 142 extends beyond the structured surfaceand is in fluid communication with an inlet (as opposed to the entirestructured surface, as shown in FIG. 14). In such embodiments, wheretroughs leading into the structured surface, rather than the entirewidth of the structured surface, are operably coupled to an inlet, thelikelihood that cells cultured on the structured surface can form atight seal and fluidly isolate the trough from the perfusion channels isincreased because, the area over which the cells need to form a tightseal to accomplish the isolation is reduced (over the trough rather thanover the entire width of the structured surface). Further, gradients ofagents introduced through the trough in such embodiments may be achievednot only from the bottom to the top of the cell culture chamber, butalso from the center to the sides of the cell culture chamber.

A microfluidic device may be made from any suitable material ormaterials and may be formed via any suitable technique. For example, amicrofluidic device, or portion thereof, may be formed via a master,such as a silicon master. The master may be fabricated from silicon byproximity U.V. photolithography. By way of example, a thin layer ofphotoresist, an organic polymer sensitive to ultraviolet light, may bespun onto a silicon wafer using a spin coater. The photoresist thicknessis determined by the speed and duration of the spin coating. After softbaking the wafer to remove some solvent, the photoresist may be exposedto ultraviolet light through a photomask. The mask's function is toallow light to pass in certain areas and to impede it in others, therebytransferring the pattern of the photomask onto the underlying resist.The soluble photoresist is then washed off using a developer, leavingbehind a protective pattern of cross-linked resist on the silicon. Atthis point, the resist is typically kept on the wafer to be used as thetopographic template for molding the stamp. Alternatively, theunprotected silicon regions can be etched, and the photoresist stripped,leaving behind a wafer with patterned silicon making for a more stabletemplate. The lower limit of the features on the structured substratesis dictated by the resolution of the fabrication process used to createthe template. This resolution is determined by the diffraction of lightat the edge of the opaque areas of the mask and the thickness of thephotoresist. Smaller features can be achieved with extremely shortwavelength UV light (˜200 nm). For submicron patterns (e.g. etch depthsof about 100 nanometers), electron beam lithography on PMMA(polymethylmetacrylate) may be used. Templates can also be produced bymicromachining, or they can be prefabricated by, e.g., diffractiongratings.

To enable simple demoulding of the master, an anti-adhesive treatmentmay be carried out using silanization in liquid phase with OTS(octadecyltrichlorosilane) or fluorinated silane, for example. Afterdeveloping, the wafers may be vapor primed with fluorinated silane toassist in the subsequent removal of the array of projections. Examplesof fluorinated silane that may be used include, but are not limited to,(tridecafluoro-1,1,2,2-tetrahydroctyl) trimethoxysilane, andtridecafluoro-1,1,2,2-tetrahydrooctyl) triethoxysilane.

By way of example and with reference to FIG. 16, an outline of a processfor forming a device or portion thereof is illustrated. As shown in FIG.16, a photoresist 410 is coated on a silicon wafer 400 (A). A photomask420, such as a chromium mask, is then placed over the photoresist (B)and the resulting assembly subjected to UV radiation. Areas of thephotoresist 410 exposed to UV are washed away (C) and the resultingstructure is etched (D) to produce the silicon master 400′, which isreplication molded with a polymer 430, such as polydimethylsiloxane(PDMS).

In some embodiments, hot embossing or injection molding may be used toform the resulting polymer. However, the silicon master may not hold upwell under conditions for such processes. In such cases, a reversesilicon master can be made and a metal, such as nickel, may be depositedon the reverse master to create a metal master for use in suchprocesses.

Any suitable material or materials may be used to form the microfluidicdevice or components thereof. For example, the device or componentsthereof may be fabricated from inorganic materials including glass,silica, silicon, metal, or the like or plastics or polymers, includingdendritic polymers, such as poly(vinyl chloride), poly(vinyl alcohol),poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride),poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers andcopolymers including copolymers of norbornene and ethylene, fluorocarbonpolymers, polystyrenes, polypropylene, polyethyleneimine; copolymerssuch as poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleicanhydride), polysaccharide, polysaccharide peptide,poly(ethylene-co-acrylic acid) or derivatives of these or the like. Thematerials for forming the devices or components or parts thereof may beselected based on desired mechanical, cell-interacting, or otherproperties for optimizing cell culture for distinct types of cells.

In various embodiments, a device is formed from two parts. For exampleand with reference to FIGS. 17A-B, the device 100 may be formed from atop part 510 and a bottom part 520. In the device 100 depicted in FIG.17A, the top 510 and bottom 520 parts are carefully aligned prior tojoining. In the device 100 depicted in FIG. 17B, the top part 510 is aplate, lid, cover, or the like that may be sealingly joined to thebottom part 520. The top part 510 may include inlets and outlets.Depending on the material used to form the top part 510 or the bottompart 520, the parts may be self sealing. Otherwise, the parts may beadhered, welded, or the like to sealingly engage.

The cell retention chamber 130 (see e.g., FIG. 17A) or a portion thereofmay be treated or coated to impart a desirable property orcharacteristic to the treated or coated surfaces. Examples of surfacetreatments often employed for purposes of cell culture include corona orplasma treatment. In various embodiments, projections or substratesurfaces are coated with a polysaccharide, such as a galactomannan, analginate, or the like, or are coated with extracellular matrix (ECM)materials, such as naturally occurring ECM proteins or synthetic ECMmaterials. The type of EMC selected may vary depending on the desiredresult and the type of cell being cultures, such as a desired phenotypeof the culture cells. Examples of naturally occurring ECM proteinsinclude fibronectin, collagens, proteoglycans, and glycosaminoglycans.Examples of synthetic materials for fabricating synthetic ECMS includepolyesters of naturally occurring α-hydroxy acids, poly(DL-lactic acid),polyglycolic acid (PGA), poly(-lactic acid) (PLLA) and copolymers ofpoly(lactic-co-glycolic acid) (PLGA). Such thermoplastic polymers can bereadily formed into desired shapes by various techniques includingmoulding, extrusion and solvent casting. Amino-acid-based polymers mayalso be employed in the fabrication of an ECM for coating a projectionor substrate. For example, collagen-like, silk-like and elastin-likeproteins may be included in an ECM. In various embodiments, an ECMincludes alginate, which is a family of copolymers of mannuronate andguluronate that form gels in the presence of divalent ions such as Ca²⁺.Any suitable processing technique may be employed to fabricate ECMs fromsynthetic polymers. By way of example, a biodegradable polymer may beprocessed into a fiber, a porous sponge or a tubular structure.

One or more ECM material may be used to coat the projections orsubstrates. For example, in embodiments, cell adhesion factors, such aspolypeptides capable of binding integrin receptors includingRGD-containing polypeptides, or growth factors can be incorporated intoECM materials to stimulate adhesion or specific functions of cells usingapproaches including adsorption or covalent bonding at the surface orcovalent bonding throughout the bulk of the materials. It will beunderstood that the type of cell or cells to be cultured in the cellretention chamber may play a role in determining which treatment orcoating, if any, may be applied to projections or other portions of thecell chamber. In many embodiments, no coating is applied to the cellculture chamber 130 or a portion thereof.

Microfluidic cell culture articles having structured surfaces asdescribed above may be used to culture a variety of cells and mayprovide important three dimensional structure to impart desirablecharacteristics to the cultured cells. Any type or combination of typesof cells (e.g., liver cells, stem cells, kidney cells, cardiac cells,neuronal or glial cells, or the like) may be cultured in suchmicrofluidic cell culture articles.

Referring now to FIGS. 18-19, general overview of methods that may beemployed using microfluidic devices as described herein are shown. Inthe method shown in FIG. 18, cells are introduced into the cellretention chamber (600), e.g. by infusing cells into the chamber via acell chamber inlet. In many embodiments, a sufficient number of cellsare introduced into the chamber to pack the chamber with cells. Whilethe number of cells to pack the chamber may vary depending on the typeof cell or cells used, between about 5000 and about 15000 hepatocytesare sufficient to pack a chamber having a volume of 0.06 mm³. In someembodiments, a number of cells less than the number of cells that wouldpack the chamber are introduced into the chamber. The cells may becultured in a manner that would allow proliferation to pack the chamberor may be cultured in manner such that the chamber does not becomepacked. The number of cells added and whether the chamber is packed maydepend on the cells being cultured, the effect to be studied, or thelike. After the cells are introduced into the cell retention chamber(600), cell culture medium may then be infused via the perfusionchannels (610). After a sufficient time (e.g., until the cells fromtight junctions, which can take about 3 days for hepatocytes), fluid maythen be infused through the trough (620) to produce a desired effect orto study the effect.

With reference to FIG. 19, the microfluidic devices described herein maybe used to determine the effect of various agents, such as smallmolecule pharmacological agents or biologic agents, on cells or theeffect of cells on the agents. Similar to the method depicted in FIG.18, the cells are introduced into the cell retention chamber of thedevice (700) and cell culture medium is infused trough the perfusionchannel(s) (710). After a sufficient time of culture, an agent may becontacted with the cells (720). The agent may be introduced via theperfusion channel(s), trough(s), or cell retention chamber. The effectof the cells on the agent, the agent on the cells, or the like may bedetermined after a suitable time of incubation of the agent with thecells (730). In some embodiments, sensors, markers, or appropriatereaders are incorporated into the device so that the effect may bedetermined. In various embodiments, fluid or cells are collected fromthe device, and the fluid is analyzed to determine the effect of thecells on the agent, of the agent on the cells, or the like. The fluidmay be collected from the perfusion channel(s), trough(s), or cellretention chamber.

As described in more detail in the Examples below, microfluidic cellculture articles having structured surfaces have been shown herein toresult in cultured hepatocytes having restored polarity and in vivo-likefunctions. In vitro cultured hepatocytes are popular for drug metabolismand toxicity studies. However, hepatocytes cultured on conventionaltwo-dimensional cell culture substrates rapidly loose their polarity andtheir ability to carry out drug metabolism and transporter functions. Toimprove the ability to maintain drug metabolism and transporterfunctions, hepatocytes have been cultured in well established in vitromodels including (i) culturing on MATRIGEL™ (BD Biosciences), an animalderived proteineous matrix, and (ii) culturing in a sandwich culturesystem between two layers of ECM such as collagen. However, such systemssuffer from significant drawbacks including the potential for phagecontamination of the human hepatocytes due to animal origin of theMATRIGEL™ or ECM materials, complex molecular compositions,batch-to-batch variations and uncontrollable coating. Culturinghepatocytes in microfluidic devices as described herein may overcome oneor more of the drawbacks of prior systems.

Any hepatocyte cell may be cultured in a microfluidic device asdescribed herein. For example, the hepatocytes to be cultured may behuman or non-human (e.g., rat, pig, etc.) hepatocytes. Examples of humanhepatocytes that may be cultured include human HepG2 cells, humanHepG2C3A cells, immortalized FaN4 cells, human primary liver cells, orthe like, or combinations thereof. The hepatocytes may be seeded in acell culture chamber of a microfluidic device at any suitable density.To pack the chamber, hepatocytes may be seeded at a density of betweenabout 100,000 cells per microliter of the chamber and about 200,000cells per microliter of the chamber. The seeding density can beoptimized, based on culture conditions and duration. For example, forlong term culture, the seeding density can be lower (e.g., 30,000 cellsto 50,000 cells per microliter of the chamber).

In some embodiments, cells capable of proliferation, such as hepG2cells, are seeded at a density lower than that which packs the chamber,and the cells may be allowed to proliferate to pack the chamber. Invarious embodiments, non-proliferating cells, such as primaryhepatocytes, are seeded at a density that packs the cell chamber.

Any suitable incubation time and conditions, regardless of the celltype, may be employed. It will be understood that temperature, CO₂ andO₂ levels, culture medium content, and the like, will depend on thenature of the cells being cultured and can be readily modified. Theamount of time that the cells are incubated in the cell retentionchamber may vary depending on the cell response being studied or thecell response desired. Prior to seeding cells, the cells may beharvested and suspended in a suitable medium, such as a growth medium inwhich the cells are to be cultured once seeded onto the surface. Forexample, the cells may be suspended in and cultured in serum-containingmedium, a conditioned medium, or a chemically-defined medium. Theoptimal culture medium for each type of cells, such as recommended byAmerican Tissue Cell Culture or other suppliers, can be used with orwithout modifications.

While not shown herein, it will be understood that microfluidic devicesas described herein may readily be multiplexed for throughput to amulti-device chip format. By way of example, such a multi-device chipformat may have a footprint of a conventional 96 well plate.

In the following, non-limiting examples are presented, which describevarious embodiments of the articles and methods discussed above.

EXAMPLES Example 1 Device Fabrication and Assembly

Four inch silicone wafers were primed with P-20 (Microprime Primer P-20,Shin-Etsu MicroSi, Phoenix, Ariz.), and a 1 um thick Shipley 1813photoresist (Rohm and Haas, Philadelphia, Pa.) was spun on the wafer at3000 rpm for 30 sec (acceleration 1000 rpm/s) and soft baked on a hotplate for 1 min at 110° C. The wafers were exposed to UV-light through achromium mask with the desired structures designed as CAD-drawing usingMA6 (Karl Suss) mask aligner. After post bake of 2 min at 80° C. thewafers were finally developed (60-100 s, MF-319, Shipley), thoroughlyrinsed with water and dried. Molds for 15 um deep troughs and 45 um deepfluidic channels and cell culture chamber were etched into the siliconeusing Plasma Therm 72 fluorine based reactive ion etcher. Afterphotoresist stripping and cleaning silicone masters were exposed totrichloro(1H, 1H, 2H, 2H)-perfluorooctyl vapor for 2 h for passivation.Polydimethylsiloxane (PDMS) replicas were produced by pouring aprecursor mixture over the whole 4″ (1:10 curing agent to prepolymer,Sylgard 184, Dow Corning, US). It was then cured at room temperature forat least 24 h, the cured PMDS was peeled of from the silicone mold tocomplete the fabrication. Room temperature curing is desired in thisprocess because it maintains high dimensional fidelity. Regular thermalcuring (65° C. or higher) produces considerable size shrinkage fromdesigned values after peeling of the PDMS structure from the master,which is not desirable for array structures because of mismatch betweenupper and lower chamber layouts. Inlet and outlet ports for mediumperfusion chamber and for cell culture chamber were punched with sharp23G style 2 needle. Upper and lower PDMS pieces were aligned using amicroscope and quickly put into contact. Reversible bonding was achievedupon conformal contact.

An image of an example silicone wafer mold resulting from the processdescribed above is shown in FIG. 20. An image of a resulting replicatedassembled device is shown in FIG. 21. Images of alternative embodimentsof devices are shown in FIG. 22A-B, which show different dimensions ofretention posts and bottom channel (or top channel) substructures. InFIGS. 21 and 22A-B, projections 144, troughs 142, retention posts 160,and perfusion channels 120, 122 are labeled for purposes of convenience.As shown in FIGS. 21, 22A and 22B, the retention posts 160, as well asthe projections 144 and troughs 142, can take be of nearly any suitableshape.

In EXAMPLES 2-6 below, a microfluidic devices made as described aboveand having a cell chambers with a bottom structured surfaces as shown inFIGS. 14D, 22A, 22B were used. The bottom structured surface had anarray of projections having a width and depth of 10 micrometers and aheight of 15 micrometers. The gap distance between projections (i.e.,the width of the troughs) was 10 micrometers. The height of the cellculture chamber was 45 micrometers and the length was 15,000micrometers.

In FIG. 21 and FIG. 22A, white lines or zig-zags were added to theimages to provide identification of troughs 142.

Example 2 Fluidic Characterization of the Microfluidic Device

To test the mass transfer inside the device between perfusion channelsand cell incubation chamber subsequent injections solutions ofSulforhodamine B (8.9×10⁻⁵ M SRB in PBS buffer) and carboxyfluoresceine(4×10⁻⁵ M in PBS buffer) dyes was performed employing a microfluidicdevice having a single inlet and outlet for both the left and rightperfusion channels. An increase of Sulforhodamine B fluorescenceintensity in the cell culture chamber was observed as a function of flowrate and time (FIG. 23A) indicating good fluidic transport across theretention barrier (posts) and between the perfusion channels and thecell chamber. Conversely, when the fluorescein (4×10⁻⁵ M in PBS buffer)was introduced via the cell chamber inlet, an increase in fluorescentintensity across the retention barrier (posts) and filling the perfusionchannels was observed (FIG. 23B). In the images shown in FIGS. 23A and23B, the left side of the image is closer to the inlet where the fluidwas introduced, and the right side of the image is closer to the outletwhere the fluid exited the device.

Cells were introduced into the main chamber of a microfluidic cellculture device via a cell chamber inlet. Approximately 5 microliters ofsuspension of primary human hepatocytes (2 million cells/ml) was used.The cells in suspension were injected into cell retention chamber at 0.5ul/min flow rate. Injection of the cell suspension was stopped once theentire cell retention chamber was packed with the cells (approximately10000 cells per device). Cell culture media was perfused though thedevice for 3 days before starting independent perfusion of bottomstructure channel. Subsequent injections of non-fluorescent (cellculture media—MFE Essential Support Medium F w/MFE Culture MediumSupplement A, #K4105.X, XenoTech LLC) and fluorescent solution(dextran-rhodamine conjugate, MW 10000, 8 mg/ml in HBSS buffer) wasintroduced via a bottom channel inlet. As shown in FIG. 24A, affixingthe cells on top of the substructures allows the introduction ofindependent flow through the substructures at the bottom of the cellretention chamber. This is evidenced by the flow of fluorescent dye inthe region of the substructures only in FIG. 24A. The fluorescent dyedid not mix or enter the left and right perfusion channels at eitherside of the cell chamber (compare FIG. 24A to FIG. 24B in whichfluorescent die was intentionally infused in the bottom and sidechannels for purposes of comparison).

In the tested device, the perfusion flow rate of the sub-structuralchannel was configured to be about 10 times lower than the perfusionflow rate of the two main side perfusion channels. Accordingly, theperfusion flow in the device may be configured to effectively mimiccomplex extracellular fluid distribution that is observed in vivo, forexample providing a gradient of bile salts as is characteristic in livertissue.

Example 3 Long Term Cell Culture in Microfluidic Devices

Incubation of human primary hepatocytes in the microfluidic devices wasperformed to demonstrate the ability of supporting phenotypically activecell population for prolonged periods of time. 5,000-10,000 primaryhuman hepatocyte cells (Cryopreserved human hepatocytes, XenoTech,Lenexa, Kans.) were plated to the microfluidic devices (via cell chamberport) and the devices were perfused with MFE cell culture medium inopen-loop mode at 1 ul/min flow rate. Cell culture media was manuallychanged daily in 96 well plates cultures. Devices were monitored dailyto track possible changes in cell morphology and health. At differenttime points the incubation was stopped and a live/dead stain (LIVE/DEADviability/cytotoxicity kit for mammalian cells from Molecular Probes,Eugene, Oreg.) was performed to monitor cell survival rate andmorphology. Images of cells packed in the device and the results oflive/dead stain are shown in FIGS. 25-26.

FIG. 25 is an image (20×) of human primary hepatocytes packed andcultured in the cell chamber of the device for 7 days. Microscopicinspection verified that the cells are packed in 3D and do not undergospreading. The hepatocytes were observed to be retained in the cellchamber (only) and did not appear to block the perfusion channels or theperfusion across the retention barriers.

FIGS. 26A-B are representative fluorescent images (A: 5×, B: 20×)showing the results of live/dead staining after 7 days of incubation.With live/dead staining, green fluorescence indicates the cells arealive and red fluorescence indicates the cells are dead. While notapparent from the black and white reproduction presented herein, nearly100% of the cells are alive (green fluorescence) after 7 daysincubation. The images presented in FIGS. 26A-B demonstrate thatmicrofluidic culture device is capable of providing effective perfusionof the assay reagents across the retention barrier and through the cellculture chamber that was packed with hepatocytes.

Example 4 The Impact of the Substructure Feature at the Bottom of theCell Culture Chamber

FIGS. 27A-B are fluorescent and brightfield images of hepatocytes packedin 3D in the cell chamber without bottom substructures (27A) and withbottom substructures (27B) taken after 7 days of incubation. In FIG.27A, the left most panel is a fluorescent image (5×) and the threerightmost panels are brightfield images at 5×, 20× and 20× (left toright, respectively). In FIG. 27B, the left most panel is a fluorescentimage (10×), the middle panel is a brightfield image (10×), and theright panel is a brightfield image (20×).

Nearly all of the hepatocytes were alive (based on green fluorescencewith live/dead staining as described above, which is not visible in theblack and white reproduction provided herein), regardless of whethercultured in a device having bottom substructures or a device having nobottom substructures. However morphologic evaluation revealed that thecells cultured on the device having no bottom substructures were nottightly fused. In contrast, the hepatocytes cultured on top of thebottom substructures of the cell chamber exhibited a more tissue-likemorphology with tightly fused cells (arrows in right most panel of FIG.27B) that show smooth edges of the 3D tissue structure as evidence ofhow well the cells are fused, resembling a tissue-like morphology.

Example 5 The Influence of Fluidics of Cells

To test the importance of perfusion flow through perfusion channels of amicrofluidic device, cells were cultured in a microfluidic device withcell culture media flow through the perfusion channels throughout andwith cell culture media present in the channels under static conditions(no continuous flow). Briefly, about 10,000 cryopreserved humanhepatocytes (XenoTech, Lenexa, Kans.) were introduced into a device. Aperfusion flow rate of 5 uL/h was used. For static conditions, cellculture media was changed manually daily. Cells were incubated for atotal of 7 days.

FIG. 28 shows three brightfield images of cells cultured under perfusionconditions. In the left panel, cells are shown in an assembled device.In the middle panel, cells are shown after the device cover was removed.The cells appear to be fused and maintained their configurationfollowing removal of the cover. In the right panel, cells are shownafter being dislodged from the device. Again, the cells maintained theirfused configuration.

FIG. 29 shows three brightfield images of cells cultured under staticconditions. In the left panel, cells are shown in an assembled device.In the middle panel, cells are shown after the device cover was removed.The cells appeared dead, individual and did not form a fused-tissue likestructure. In the right panel, cells are shown after being dislodgedfrom the device. Again, the cells appeared dead, individual and did notform a fused-tissue like structure.

FIG. 30 shows two brightfield images of cells cultured on a 96 wellplate having the structured bottom surface of the microfluidic devicereplicated on the bottom surface of the well. About 60,000 cryopreservedhuman hepatocytes (XenoTech LLC, Lenexa, Kans.) were seeded per well inMFE Essential Plating Medium F (#K4000). After 24 h incubation, themedium was changed to MFE Essential Support Medium F w/MFE CultureMedium Supplement A (#K4105.X, XenoTech LLC). During the incubationmedium was changed daily. The cells were incubated for 7 days. The imageon the left is of cells cultured on plasma treated PDMS substrate. Theimage on the right is on cells cultured on non-treated PDMS. In bothcases, the static cell culture conditions did not support a 3Dtissue-like cellular structure. Hepatocytes cultured on structured PDMSsurface in 96 well plate under static conditions formed a monolayerculture and did not preferentially recognize structured regions relativeto flat regions.

The microfluidic device provides multiple independent perfusion channelsfor cells culture in continuous media (fluid) flow. Fluid perfusionbased on the design dimensions mimics the hepatic circulation providingefficient continuous transport of gas and nutrients to the hepatocytesand removal of metabolites or cellular waste. The microstructured lowerflow channel at the bottom of the cell chamber allow for independentperfusion from the other two perfusion channels of the device. Themultiple perfusion channels effectively transport media (nutrients),assay reagents and cellular waste, thereby, maintaining a viable cellculture for an extended period of time. Also, the dynamic cell cultureconditions influence the formation of 3D cells that are tightly fusedinto a tissue-like cellular structure without the addition of animalderived or synthetic matrices or coagulants that remains intact whendislodged from the device (FIG. 28).

Static culture conditions in the device resulted in individual cellsthat died during incubation and readily dispersed when the device wasdissembled (FIG. 29). Likewise, static cell culture conditions with astructured PDMS surface in 96 well plate, a replication of the bottom ofthe cell chamber of the device, did not support a 3D tissue-likecellular structure. Hepatocytes cultured on structured PDMS surface in96 well plate under static conditions formed a monolayer culture and didnot preferentially recognize structured regions relative to flat regions(FIG. 30).

Example 6 Restoration of Membrane Polarity and Hepatocyte SpecificFunction

Hepatocytes, in vivo, are supported in three dimensional conformation bya combination of extra cellular matrix (ECM) and other non-parenchymalcells. In conventional in vitro 2D cell culture format primaryhepatocytes dedifferentiate rapidly because of limited cell-cellinteraction and the inability to restore in vivo-like cellularorganization. The maintenance of differentiated functions of primaryhepatocytes is dependent on the restoration of morphological structureand membrane polarity. The metabolic functions of primary hepatocyteshave been clearly correlated to the polarity of hepatocytes induced bydifferent culture configurations. Therefore, restoration of hepatocytepolarity is important in the maintenance of hepatocyte function.

As mentioned above, the substructures at the bottom of the cell chamberprovide a lower flow channel through these microstructures whichprovides independent perfusion. This design feature influences theformation of tightly fused, tissue-like cellular structures. The 3D cellculture morphology promotes the restoration of cell membrane polarity(FIGS. 31-32), and the restoration of hepatocyte specific function, suchas transport function (FIG. 33) without the addition of animal derivedor synthetic matrices or coagulants. Cell membrane polarity is evidencedby the extended formation of bile canaliculi structures in 3D shown bythe expression of the bile canalicular marker, MRP2, and gap junctionprotein, Connexin 32, via immunostaining in FIGS. 31-32. Briefly, afterseven day perfusion culture inside microfluidic device, cells were fixedwith 3% paraformaldehyde in PBS, permeabilized with Triton X-100 (1% inPBS) and incubated with a mixture of primary antibodies (1:100 dilutionby blocking buffer, mouse anti-Connexin 32 unconjugated monoclonalantibody, 25 ug/ml; Rabbit anti-MRP2, polyclonal antibody, 9 ug/ml,Abcam) overnight at 4° C. After the incubation, the sample was washedwith 0.1% Tween 20 in PBS and incubated with secondary antibodiesconjugated to FITC (494/518 nm) and Cy3 (550/570 nm) fluorescent labels.Unbound antibodies were washed out with washing buffer (3×200 uL) andthe sample was covered with 20 uL of Vectashield mounting solutionsupplemented with fluorescent DAPI stain to stain the cells' nuclei.

In conventional 2D cell culture the expression of MRP2 protein whenpresent is observed as tiny unconnected dots between some cellsillustrating the limited formation of bile canaliculi structures (seeFIG. 33A, in which hepatocytes were cultured on a conventional 96 wellplate). After 7-day culture on conventional 96 well plate, cells wereincubated for 10 min with MPR2 substrate (5 μM 5-(6) carboxy-2′7′dichlorofluorescein diacetate solution in cell culture media).Carboxy-2′7′ dichlorofluorescein diacetate was absorbed by the cell andmetabolized. The metabolites are actively excreted by MRP2 protein intobile canalicular structures. Transposition of fluorescein metaboliteswas monitored by fluorescent microscopy (494/518 nm), thus, dynamicfunctional stain of bile canalicular structures inside the cellaggregates was obtained.

The MRP2 protein is also responsible for transport function from thecells into the bile canaliculi structure. Transport function, in recentyears, is often referred to as Phase III of drug metabolism in the liverand is critical for removal of drug metabolites or the active transportof drug compounds into cells. FIG. 33B presents one image of the dynamicassay where fluorescein diacetate was passively absorbed by thehepatocytes and actively transported via MRP2 transporter protein intoextended bile canaliculi structures demonstrating hepatocyte specificfunction in the microfluidic device. Briefly, MPR2 substrate (5 μM 5-(6)carboxy-2′7′ dichlorofluorescein diacetate solution in cell culturemedia) was perfused though the device for 10 min at 1 uL/min flow rate.Transposition of fluorescein metabolites can be monitored by fluorescentmicroscopy (494/518 nm), thus, dynamic functional stain of bilecanalicular structures inside the device was can be obtained. Thisfunction is largely driven by restoration of polarity via cell-cellsignaling and the formation of tightly fused, tissue-like, 3D cellularstructure. To our knowledge, the formation of such extended bilecanaliculi structure in 3D (restoration of membrane polarity) and thedemonstration of transport function of human primary hepatocytes in amicrofluidic device has not be shown or reported before.

In summary, the present disclosure describes a microfluidic devicedesign and methods for functional maintenance of cells in highlydifferentiated state in vitro. The ability of cells to support its invivo functions while being cultured in vitro system have high importancein tissue engineering applications and evaluating therapeuticcandidates. The described cell culture systems may be used to supportlong term cell cultures, to promote restoration of in vivo like cellularorganization that increase phenotype specific activity of cultured cellsthus providing physiologically relevant information for cell-basedassays.

Thus, embodiments of MICROFLUIDIC DEVICE FOR CELL CULTURE are disclosed.One skilled in the art will appreciate that the cell culture apparatusesand methods described herein can be practiced with embodiments otherthan those disclosed. The disclosed embodiments are presented forpurposes of illustration and not limitation.

What is claimed is:
 1. A cell culture apparatus comprising: a cellretention chamber having a first structured surface, wherein thestructured surface includes a major surface from which a plurality ofprojections extend into the chamber, wherein the plurality ofprojections are arranged to suspend cells cultured in the chamber abovethe major surface; and a first perfusion channel (i) configured to carrya cell culture medium and (ii) forming a plurality of openings incommunication with the cell retention chamber, the openings configuredto prevent cells from the retention chamber from entering the perfusionchannel.
 2. A cell culture apparatus according to claim 1, wherein eachof the plurality of projections has a cell contact surface configured tocontact one or more of the cells, wherein the cell contact surface has adiametric dimension less than 15 micrometers.
 3. A cell cultureapparatus according to claim 2, wherein the cell contacting surface ofeach of the projections has a diametric dimension of between 5micrometers and 10 micrometers.
 4. A cell culture apparatus according toclaim 1, wherein each of the projections extend from the major surfaceto a height of between 5 micrometers to 25 micrometers.
 5. A cellculture apparatus according to claim 1, wherein the distance betweenneighboring projections is less than 15 micrometers.
 6. A cell cultureapparatus according to claim 1, wherein the distance between neighboringprojections is between 5 micrometers and 10 micrometers.
 7. A cellculture apparatus according to claim 1, wherein a trough is formedbetween the plurality of projections, wherein the trough extends thelength of the structured surface.
 8. A cell culture apparatus accordingto claim 7, wherein the apparatus further comprises an inlet in fluidcommunication with the trough.
 9. A cell culture apparatus according toclaim 8, wherein the apparatus further comprises an outlet in fluidcommunication with the trough, and wherein the outlet is the same ordifferent from the inlet.
 10. A cell culture apparatus according to anyof the preceding claims, further comprising a second perfusion channelforming a plurality of openings in communication with the cell retentionchamber, the openings being of a dimension configured to prevent cellsfrom the culture chamber from entering the second perfusion channel. 11.A cell culture apparatus according to claim 10, wherein the first andsecond perfusion channels are on generally opposing sides of the cellretention chamber.
 12. A cell culture apparatus according to claim 10,wherein the cell retention chamber has a width of between 80 micrometersand 110 micrometers.
 13. A cell culture apparatus according to claim 10,wherein the cell retention chamber has a height of between 40micrometers and 50 micrometers.
 14. A cell culture apparatus accordingto claim 10, wherein the first perfusion channel has a height of between30 micrometers and 50 micrometers and has a width of between 30micrometers to 50 micrometers.
 15. A cell culture apparatus comprising:a cell retention chamber having a first structured surface, wherein thestructured surface includes a major surface from which a plurality ofprojections extend into the chamber, wherein each of the plurality ofprojections has a cell contact surface opposing the major surface,wherein the cell contact surface has a diametric dimension of less than15 micrometers, each of the projections extend from the major surface toa height of greater than 5 micrometers, and wherein the distance betweenneighboring projections is less than 15 micrometers, wherein thestructured surface includes a trough, at least a portion of which isformed between neighboring projections, wherein the trough extends thelength of the structured surface; a trough inlet in fluid communicationwith the trough; a chamber inlet in fluid communication with the cellretention chamber; and a first perfusion channel (i) configured to carrya cell culture medium and (ii) forming a plurality of openings incommunication with the cell retention chamber, the openings configuredto prevent cells from the retention chamber from entering the perfusionchannel.
 16. A cell culture apparatus according to claim 15, wherein thetrough is configured to be at least partially isolated from the chamberwhen cells are cultured in the chamber.
 17. A cell culture apparatusaccording to claim 2, wherein each of the projections extend from themajor surface to a height of between 5 micrometers to 25 micrometers.18. A cell culture apparatus according to claim 2, wherein the distancebetween neighboring projections is less than 15 micrometers.
 19. A cellculture apparatus according to claim 2, wherein the distance betweenneighboring projections is between 5 micrometers and 10 micrometers. 20.A cell culture apparatus according to claim 2, wherein a trough isformed between the plurality of projections, wherein the trough extendsthe length of the structured surface.